Isolated population of plant single cells and method of preparing the same

ABSTRACT

This invention is a method of minimizing the variation of cell growth and production through homogeneous cell line development. To be more specific, it is the method of isolating and proliferating single cell clone from the procambium to promote the stability of the plant-derived biologically active substances production by solving the problems of decrease in cell growth and the productivity during the long term culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/063,929 filed in the U.S. Patent & Trademark Office on Feb.15, 2008 entitled “Stability of Secondary Metabolite Mass ProductionThrough Synchronized Plant Cell Cultures” in the name of Young Woo Jin,which was filed under the provisions of 35 U.S.C. §371 and claims thepriority of International Patent Application No. PCT/KR2006/001544 filedon 25 Apr. 2006, which claims priority to Korean Patent Application No.10-2005-0103445 filed on 31 Oct. 2005, which are all hereby incorporatedby reference in their entirety.

TECHNICAL FIELD

Plant has been used very importantly not only as our food supply butalso as the source of extensive chemical substances including,pharmaceuticals, fragrances, colors, agricultural chemicals and dyesetc. Biologically active compounds that are produced from plants aremostly secondary metabolites. There is a greater interest on thesecondary metabolites, such as alkaloid, allergen, amino acid,anthraquinone, antileukaemic agent, antimicrobial agent, antitumoragent, antiviral agent, enzyme, flavonoids, insecticide, opiate,perfume, pigment, vitamin, and polysaccride etc., because most of themwork as physiologically active substances. According to Zhong (2002),there are about 100,000 known plant secondary metabolites and more than25% of the medicine that are practically used is plant-derivedsubstances. Every year, novel secondary metabolites are discoveredcontinually.

In the method of obtaining these metabolites, there are many problemssuch as difficult chemical synthesis in spite of the recent astonishingdevelopments of the organic chemistry, demolition of the nature due toexploitation and environmental pollution and changes of the content ofmetabolites and increase of the production cost depending on the cultureconditions, like season, region and climate. Therefore, there are ongoing active attempts to produce secondary metabolites through in vitroculture technique which has advantages of controlling the adequateexternal environmental conditions and producing on a large scale even ina small space.

BACKGROUND ART

According to KR patent 0130100, production of biologically activesubstances through plant cell culture has more advantages than directextraction from the plant. Plant cell culture is considered as anoptimal method for continual production which is not influenced byenvironment and for solving the pending problems like destruction ofecology.

Nail & Roberts (2004), however, indicated slow growth rate and lowproductivity of the plant cell culture for the secondary metaboliteproduction. To solve this problem, there are studies of the optimizationof the media, culture conditions, process and elicitation for higherproductivity etc. (Zhong 2002). In the International patent WO93/17121,various media was used to culture diverse Taxus for the increase in cellgrowth rate and paclitaxel productivity. Based on the results of theexperiments, elicitation conditions for paclitaxel mass production wasindicated. Despite the improvements to the production of valuablesecondary metabolites, variability is still a major issue for theproduction of paclitaxel from Taxus and other valuable substances fromnumerous plant systems.

Production of secondary metabolites through large scale plant cellculture is commercially possible only when there is a stable maintenanceof rapid cell growth and high metabolite production during long termculture. The ability of the cell lines that could produce distinctmetabolites are not stable which cause the cell lines to lose theirinitial productivity through subcultures; it is not too much to say thatsuccess and failure are depended on how we overcome these problems.

In plant cell culture, although the cells are derived from one plant,metabolite productivity of each cell line is different and unstable.Therefore, establishing the cell lines that have high productivity andgenetic stability is most important than anything else.

Cell Lines Derived from Single Cells & and Multiple Cells

Plant cell lines derived from single cells have lower variability thanthe cell lines derived from multiple cells; this results in higherproductivity. In preceding inventions, stem, root, seed, needle and leafwere used as the best explants for cell line induction. These stem,root, seed, needle and leaf are tissues that are composed of the cellswith distinct functions and morphology. Callus, cell lines derived fromthese tissues is not of one kind. Therefore, there are limitations onthe attempts to reduce the productivity variation of the callus derivedfrom the tissues consisted of multiple cells.

Cell Aggregation

One of the distinguishing characteristics of plant cell culture is cellaggregation. According to the patent 0364478, diameter of the plant cellis 30-300 μm which is about 30 times bigger than the animal cell.Because plant cell walls have natural tendency to adhere together, it isnot possible to obtain suspension which consists only of dispersedsingle cells. The proportion and the size of cell aggregates varyaccording to plant variety and the medium in which the culture is grown.Nail & Roberts indicated that cell aggregation leads to a difference inlocal environment between interior and exterior of the cells, which canresult in culture heterogeneity and ultimately leads to changes ingrowth and metabolism.

The purpose of suspension culture is to obtain pure single cells. Toaccomplish this objective, filtration, maceration and protoplast cultureby using enzyme were used. However, filtration and maceration do notprovide complete pure single cells. Protoplast culture technique whicheliminates the cell wall is the most reliable method for generatingsingle cells, but the enzyme used for the protoplast culture cause cellwall damages or breakages that result in the change of cell physiology.Moreover, hydrophobic secondary metabolites such as paclitaxel can bestored in the cell wall, so the changes in the cell wall have profoundrelationship with productivity.

Also, cell aggregation has long been a major obstacle to the accuratemeasurement of cell growth by number and to biochemical assays toindividual cells. According to Nail & Roberts (2004), if single cellculture is possible, it will readily provide faster information aboutthe behavior of cell units in the culture such as biosynthesis, storage,and degradation etc. of secondary metabolites.

Dedifferentiation

The dedifferentiated cell line, which is callus, shows great variabilityin the production of secondary metabolites due to somaclonal variation.Callus derived from the permanent tissues such as leaves, stem, root andseed that are composed of the cells with distinct functions andmorphology usually show dramatical changes even on slightly differentmicro-environments because it is a secondary meristem formed bydedifferentiation. Due to this sensitivity, Hirasuna et al. (1996)investigated to identify the cell culture conditions, especially initialcell density, subculture interval and temperature, and to maintain themas precisely as possible.

Scale Up

In order to produce secondary metabolites through plant cell culture forcommercialization, scale up is essential. Bioreactor has been appliedfor mass production after many patents and articles were published,reporting about successful production of metabolites through cellculture in a laboratory scale. According to patent 0290004, applicationof bioreactor for mass production provides very different cultureenvironment from the flask in a laboratory scale which results in thedecrease in growth rate and productivity and change in the metabolites.When the bioreactor is applied for mass production, changes in growthrate, productivity and metabolites have become problems incommercialization of biologically active substances through cellculture. In the scale up of plant cell cultures, bioreactor whichreceives the air through exterior power or the bioreactor with impellerby considering the efficiency of the mixing and aeration are preferred.However, cell viability decreases abruptly in the bioreactor becauseplant cells are weak for shear. Therefore, a method to reduce shear isnecessary. The cause of the shear sensitivity of the plant cell isexplained by its large size, rigid cell wall, aggregation and extensivevacuolate (Yokoi, et al, 1993). To solve these problems in thebioreactor, a low shear generating bioreactor was investigated in thepast by controlling its agitating speed and modifying the impeller type.However, it still bears negative results because the cell lines couldnot overcome the differences of the microenvironment.

Cryopreservation

Cryopreservation allows the long term cell maintenance by ceasing mostof the metabolism of the cells in the extremely low temperature. Itsignifies the recovery of the cells without genetic, characteristics andbiosynthetic variation after cryopreservation. By usingcryopreservation, lost of the cells from contaminations could beeliminated and the genetic variation in the continuous cell lines couldbe minimized. In cGMP, the preservation of the cell lines for a longperiod is mandatory for the stable supply of raw materials. Usually,cultured animal cells could undergo cryopreservation for many years, butthe similar cryopreservation technique is much more challenging forcultured plant cells. Cultured plant cells are heterogeneous and showdiversity in physiology and morphology. Therefore, plant suspensioncells require many processes for cryopreservation and inadequatecryopreservation could cause variability.

Conditioning Factors

Kim et al. (2000) demonstrated that cell division can be stimulated ifsome media from actively dividing cultures was added to the culturesthat lost cell division ability. In the production of anthocyaninthrough rose suspension culture, the productivity increased when somemedia of strawberry suspension culture was added to the rose suspensionculture. In this way, the factors that were produced and secreted fromthe cultured cells to stimulate the cell growth or the production of thesecondary metabolites are called conditioning factors. Yet, theseconditioning factors have not been identified concretely and there areonly some understanding of conditioning factors acting as chemicalsignals for the cell growth and metabolite production. Also, there arefew reports on the potent substances, such as phosphates and calmodiumwhich could be considered as conditioning factors. Conditioning factorscan be supplied through conditioned media or helper cells.

Perfusion Cultivation

Among the cell culture methods, there is a batch cultivation involvingthe inoculation of the cell and the media together in the beginning andno further nutrition supplementation. Also, there is a continuouscultivation, involving the supplementation of the new media as the spentmedia that contains metabolites is retrieved simultaneously at aconsistent speed during the culture period for the prevention ofnutrition depletion.

Batch cultivation is difficult in the commercial level due to its lowproductivity. Among the continuous culture methods, perfusioncultivation is receiving much attention these days. In perfusionculture, the cells are remained in the bioreactor, and new media issupplied as the spent media that contains metabolites is retrieved.

According to Zhang et al. (2000), elicitation is one of the mosteffective ways to promote the secondary metabolites production in cellculture. Elicitation encourages secondary metabolite synthesis, but itinduces cell growth inhibition and the rapid decrease in the cellviability. Hence, secondary metabolite synthesis by elicitation could bemaintained only for a short period and it is very limited. As Wang etal. (2001) presented, perfusion cultivation is a strategy to minimizethese negative effects by elicitation and to maximize the productivity.

Wang et al. (2001) and Wu & Lin (2003) reported as follows. Secondarymetabolites that are produced by elicitation are stored inside of thecell (vacuole or cell wall) or released outside of the cell (media).During the process of culture, releasing secondary metabolites from thecell and removing it from the media could bring easier purification andcould diminish the feedback inhibition of biosynthesis and degradationand conversion of the products. Therefore, by retrieving the spent mediaand supplying with a new media, secretion of internal and externalmetabolites could extend the viability and biosynthesis of the cells.And it could remarkably increase the productivity.

Storage and the secretion of secondary metabolites showed greatdifferences depending on the cell lines. Taxus media cell line(Wickremesinhe and Arteca 1994) did not excrete any. Consequently,establishing the cell line that has outstanding secretion ability isrequired.

Cambium Culture

Cambium is a lateral meristem that is located on the lateral side of theplant. In the gymnosperm and woody dicotyledon plants, there is ahypertrophic growth due to the continual activity of the cambium; as aresult, giant plants having more than 11,000 years of the growth ringsexist. In genetics, meristems could be classified as primary andsecondary meristem. Primary meristem represents the meristem that formsduring embryogenesis and participates in the plant growth after seedgermination. Secondary meristem represents the meristem that is formedby dedifferentiation of the plant permanent tissue. Cambium is a primarymeristem with meristematic continuity derived from the procambiumwithout the intervene of the permanent tissue.

Growth of this primary meristem is indeterminate and could be continuedif the conditions are given. Therefore, cambium culture has been usedfor rapid mass propagation of the cells.

In the preceding studies, cambium explants were prepared as follows:after the bark was peeled off, two longitudinal cuts, approximately 1 mmdeep in order to reach the xylem, were made into the woody stem at aninterval of 5 mm. They called these explants ‘cambium’, which wasconstituted of part of the phloem, cambium and a small chip of xylem(Jouira et al., 1998).

It is reasonable to say that cells which are induced by the method asmentioned above are not the sole origin of cambium, but of multipletissues, which can be solemnly distinguished anatomically such asphloem, cambium and xylem. Thus, we could indicate that the methodmentioned above is not the ideal technique to separate only the cambiumelaborately from the various tissues that constitute the stems. Acreative method to separate only the cambium or procambium from thevarious tissues of stems has been in demand.

DISCLOSURE Technical Problem

The objective of this invention is to generate the method to producesingle cell clone by separating and culturing only the procambium fromthe twig. To put it concretely, the goal of this invention is to solvethe variation in plant cell culture and to generate stable productionmethod for plant biologically active substances by separating theprocambium purely through combining the methods of cell andphysiological chemistry separation to the preceding physical separationmethod that utilizes the scalpel.

Another purpose of this invention is to generate stable cellproliferation by separating and culturing only the procambium from theTaxus twig and to generate the method of paclitaxel production.

Technical Solution

To achieve the above objectives, in one aspect, the present inventionprovides a method for isolating plant procambium-derived single cellclone, the method following: (a) preparing and then sterilizing theplant tissue; (b) collecting the tissue containing procambium from thesaid sterilized plant tissue; (c) culturing the said tissue containingprocambium, and thereby inducing a procambium layer which isproliferated from procambium, and a callus layer which is derived fromregions except procambium and proliferated in an irregular form; and (d)collecting the single cell clone by isolating the said procambium layerfrom the said callus layer.

Preferably, the step (c) comprises culturing the said tissue in mediumcontaining auxin. And in a preferred embodiment, the medium contains 1˜3mg/L of the auxin.

In another aspect, the present invention provides a single cell cloneinduced from plant procambium, the single cell clone has the followingcharacteristics: (a) above 90% cells in suspension culture exist assingle cells; (b) having multiple vacuoles morphologically; (c) growingfaster than the cell line derived from regions except procambium of thesame plant origin, and culturing stably for a long time; (d) having lowsensitivity to shear stress in the bioreactor; and (e) being innatelyundifferentiated.

Preferably, the plant is the genus Taxus. And in a preferred embodiment,the genus Taxus procambium-derived single cell clone has an ability ofreleasing 404˜1077 times more paclitaxel than the cell lines derivedfrom regions except procambium of the same plant origin.

In still another aspect, the present invention provides a method forproducing plant-derived biologically active substances, the methodcomprising the steps of: (a) producing the active substances byculturing the above single cell clone; and (b) collecting said activesubstances. Preferably culturing of the step (a) comprises retrievingthe media used in culturing of said single cell clone culture and thensupplying with a new media.

In a preferred embodiment, the single cell clone is the genus Taxusprocambium-derived single cell clone, and the compound is paclitaxel. Inthis case, the media may further contain one or more materials selectedfrom the group consisting of methyl jasmonate, phenylalanine andchitosan.

And, the present invention provides a method for preserving a plant cellline, the method comprises cryoperservating single cell clone derivedfrom plant procambium, which are isolated by the above method.

Advantageous Effects

According to the methods of this invention, it is possible to culturesingle cell clone that has the meristematic continuity of primarymeristem without going through dedifferentiation by precisely separatingonly the procambium from various tissues of woody plant twig or stem.Cell line of this invention allows stable production of biologicallyactive substances due to less change in the cell growth rate and growthpattern during the long term culture. It is also optimal for the massproduction in commercial level because it is less sensitive to shear inthe bioreactor compared to the cell lines derived from the precedingtechniques due to less aggregation and multiple vacuoles.

Metabolite activation can be stimulated by supplementing conditioningfactors to this cell line and cell vitality and biosynthesis can beextended as the cells releasing considerable amount of production intothe extracellular media through perfusion culture. High recovering rateafter cryopreservation due to homogeneity and division ability of thiscell line devises the establishment of cell bank. Through thisinvention, close relationship between homogeneity of the cultures andvariation of secondary metabolites are confirmed, and the method of thisinvention could develop the strategy for commercialization as itcontrols and reduces the variability of diverse biologically activesubstance production.

DESCRIPTION OF DRAWINGS

FIG. 1 is the part which was separated during the induction of singlecell clone from the procambium (A: Macrography of twig after 30 days ofcultivation—the procambium (bottom) is separated by callus cells derivedfrom tissue consists of a primary phloem, cortex and epidermis; B:Single cell clone derived from procambium after 35 days of cultivation;C: Callus derived from tissue consists of a primary phloem, cortex andepidermis after 40 days of cultivation; D: Callus derived from embryo orneedle after 50 days of cultivation).

FIG. 2 is the growth rate expressed by the total biomass production ofthree different cell cultures derived from procambium, embryo and needleof T. cuspidate in 22 months (The subculture interval ranged between 14days).

FIG. 3 is the image of cell aggregation of the cultures derived from twodifferent tissues (A & B: embryo or needle; C & D: procambium) of T.cuspidata (A: Large cell aggregates, size higher than 1.5×10² μm; C:single cell population; D: cell presenting a high density of vacuole.).

FIG. 4 is the effects of elicitors and their combinations on paclitaxelproduction in T. cuspidata (single cell clone from procambium)suspension cultures (Conditioning factors were incorporated to zero-daycultures. Symbols: C, control; Chi, 50 mg/L Chitosan; Phe, 0.1 mMPhenylalanine; MJ, 100 μM Methyl jasmonate; Com (Combined-elicitedcultures), the combination of 50 mg/L Chitosan, 0.1 mM Phenylalanine and100 μM Methyl jasmonate.).

FIG. 5 is the effect of conditioning factors on paclitaxel production inT. cuspidata (single cell clone from procambium) suspension cultures(Elicitors (50 mg/L Chitosan, 0.1 mM Phenylalanine and 100 μM Methyljasmonate) were incorporated to 14-day-old cultures. Symbol: CF,conditioning factors.).

MODES OF THE INVENTION

Practical examples of the invention are explained below. Induction andproliferation method of single cell clone from the procambium is notonly utilized in paclitaxel production system but it may also beutilized in all plant secondary metabolite production system. Thefollowing examples are offered by way of illustration, not by way oflimitation.

Practical Example 1 Preparation of Plant Materials and Isolation ofProcambium

Seed, needle, twig of the yew tree were collected. After collecting thematerials, they were deposited in the solution of 100 mg/L ofantioxidant, ascorbic acid (L-ascorbic acid, DUCHEFA, The Netherlands)immediately and transferred and preserved. They were surface sterilizedby considering the morphology and physiological characteristics of thematerials.

-   -   {circle around (1)} Seed: After sterilizing the seeds with 70%        ethanol for one minute, they were immersed in 1% Clorox solution        for 48 hours and were washed 3 to 4 times with sterile water.        Next, embryo was separated from the seed in the solution of 0.5%        PVP (poly vinyl pyrrolidone, DUCHEFA, The Netherlands) and 50        mg/L of ascorbic acid (L-ascorbic acid, DUCHEFA, The        Netherlands), and 70 mg/L of citric acid (DUCHEFA, The        Netherlands) and cultured on the callus induction media.    -   {circle around (2)} Needle and twig: After 24 hours of treatment        with the solution containing 1% Benomyl (Dongbu Hannong        Chemical, Korea)+1% Daconil (Dongbu Hannong Chemical, Korea)+1%        Streptomycin sulphate (DUCHEFA, The Netherlands)+0.1% Cefotaxime        sodium (DUCHEFA, The Netherlands), needles and twigs were rinsed        with tap water for 30 seconds to remove the remaining chemical        substances and phenolic compounds. After sterilizing them with        70% ethanol (DC Chemical, Korea) for one minute, 30% hydrogen        peroxide (LG Chemical, Korea) for 15 minutes, 1% CLOROX solution        for 15 minutes, 3% CLOROX solution for 5 minutes in order, they        were washed 3 to 4 times with distilled water. To prevent the        oxidation, both ends of the needle were cut in the solution of        0.5% PVP, 50 mg/L ascorbic acid and 70 mg/L citric acid and        cultured on the callus induction media.    -   {circle around (3)} Procambium preparation from an apex of the        twig: By holding the xylem which is the center region of an apex        in the twig with the tweezers, phloem and cortex and epidermis        tissues including the procambium were peeled off. This peeled        tissue that contained procambium were laid on the media;        procambium was allowed to touch the surface of the media.

Practical Example 2 Induction of Single Cell Clone from the IsolatedProcambium

After 4 to 7th day of the culture, cell division of the procambium wasobserved and on the 15^(th) day of the culture, callus was beginning toform from the layer consisted of the phloem and cortex and epidermisthat were the upper part of the procambium. On the 30^(th) day of theculture, the procambium began to be separated from the upper layertissue that contained the phloem and cortex and epidermis; after thesetwo layers were completely separated naturally, they were culturedindividually on different petri dishes (FIG. 1).

For the purpose of cell and callus induction, universally known media ofthe plant cell and tissue culture could be used: e.g. mB5 (modifiedGamberg's B5 medium), MS (Murashige & Skoog medium), WPM (Lloyed &McCown), SM (schenk & Hildebrand medium), LP (Quoirin & Lepiovre).Application of all these media is possible. Various additives could besupplemented and components of the media could be reduced or eliminatedas the need arises. Among them, the most appropriate media was mB5. Thecontents of mB5 are described in the following Table 1.

TABLE 1 Table 1. Cell line induction & maintenance medium in Taxus spp.Contents Composition (mg/L) Inorganic salts KNO₃ 2500 (NH₄)₂SO₄ 134MgSO₄•7H₂O 121.56 MnSO₄•4H₂O 10 ZnSO₄•7H₂O 2 CuSO₄•5H₂O 0.025 CaCl₂•2H₂O113.23 KI 0.75 CoCl₂•6H₂O 0.025 NaH₂PO₄•H₂O 130.44 H₃BO₃ 3 Na₃MoO₄•2H₂O0.25 FeNaEDTA 36.7 Vitamin Myo-Inositol 200 Thiamine-HCl 20 Nicotinicacid 2 Pyridoxine-HCl 2 L-ascorbic acid 50 Citric acid 75 Amino acidL-aspartic acid 133 L-arginine 175 Glycine 76 Proline 115 Hormonea-Naphtalene acetic acid 2 Sucrose 10,000 Activated charcoal 100 Gelrite2,000

The cultures were grown on the media that was supplemented with a plantgrowth regulator, auxin (1-3 mg/L) in the dark at 25+1° C.

Procambium was composed of homogeneous cells, so its cell division wasuniform and proliferation occurred in the form of a plate. On the otherhand, the tissue containing the phloem and cortex and epidermisproliferated in irregular form because there was a discrepancy of celldivision due to the composition of many kinds of cells. There was aself-split of the layer in between the procambium and the tissuecontaining phloem and cortex and epidermis (FIG. 1). Procambium washomogeneous and the tissue containing phloem and cortex and epidermiswas heterogeneous, so the self-split of the layer seemed to be theresult of different division rate.

After 15^(th) day of the culture, calli were formed on the explants ofembryo and needle that are composed of heterogeneous cells bydifferentiation and these calli proliferated in irregular forms due tothe different division rate of various cells just like the tissue thatcontained phloem and cortex and epidermis. (FIG. 1)

Practical Example 3 Establishment of Long Term Culture

Among the calli, white and friable parts that had good growth rate weresubcultured onto the new media every 14 days. Growth rate of the embryoand needle-derived cultures was very unstable and it often showed thetendency of browning. On the contrary, growth rate of procambium-derivedcultures was fast and there was no color change of the cultures.Therefore, it was possible to select the stable cells.

After six months of the culture, most of the embryo and needle-derivedcultures had yellow or light brown color and aggregation formed.Procambium-derived cultures had white-yellow color and were maintainedas single cells or small cell clusters. Growth rate of the cultures thatturned brown and formed aggregation slowed down and the cultures diedeventually because of the phenol chemical substance that they excreted.

According to this inventor, maintenance and mass proliferation of theembryo and needle-derived cultures was difficult after 6 months, butprocambium-derived cultures were maintained stably for more than 20months of the long term culture without any variation in the rate ofcell growth, growth pattern and aggregation level (FIG. 2). In otherwords, variability appeared in growth pattern, depending on thehomogeneity and heterogeneity of the initial plant materials.

Practical Example 4 Establishment of Cell Suspension Culture

The embryo and needle-derived and procambium-derived cultures werecultured individually in the flask containing the liquid media (Table2).

TABLE 2 Table 2. Suspension medium in Taxus spp. Contents Composition(mg/L) Inorganic salts Ca(NO₃)₂ 471.26 NH₄NO₃ 400 MgSO₄•7H₂O 180.54MnSO₄•4H₂O 22.3 ZnSO₄•7H₂O 8.6 CuSO₄•5H₂O 0.25 CaCl₃•2H₂O 72.5 K₂SO₄ 900Na₂MoO₄•2H₂O 0.25 H₃BO₃ 6.2 KH₂PO₄ 170 FeNaEDTA 36.7 VitaminMyo-Inositol 200 Thiamine-HCl 20 Nicotinic acid 2 Pyridoxine-HCl 2L-ascorbic acid 50 Citric acid 75 Amino acid L-aspartic acid 133L-arginine 175 Glycine 76 Proline 115 Hormone a-Naphtalene acetic acid 2Sucrose 30,000

They were cultured on the 100 rpm rotating shaker in the dark at 25±1°C. With the two weeks of subculture interval, cultures were allowed tomaintain high vitality continuously as exponential growth phase.

Aggregation level which is the main cause of the variation of cellproductivity was measured. Cell aggregate quantification was measuredwith the biological microscope (CX31, Olympus, Japan). The result of theexperiment described above is on Table 3.

TABLE 3 Type of cell aggregates of Taxus long-term cultures Large cellModerate cell Small cell Single cell aggregates aggregates aggregatespopulation Explant source 60 ± 3.2% 30 ± 3.3%   7 ± 0.6%   3 ± 0.9%embryo, needle 0 0 7.4 ± 0.8% 92.6 ± 0.8% procambium Large cellaggregates, size higher than 1.5 × 10³ μm; Moderate cell aggregates, 1 ×10³ μm; Small cell aggregates, 4 × 10² μm < size < 1 × × 10³ μm

In case of the suspension of the embryo and needle-derived cultures,about 60% had cell aggregation size more than 1.5 mm but in thesuspension of procambium-derived cultures, 92.6% of the cells werecultured as single cells.

Practical Example 5 Scale Up

Embryo and needle-derived and procambium-derived cultures were culturedin 3 L airlift bioreactor (Sung-Won SciTech, Korea) in the dark at 25±1C°.

In case of the embryo and needle-derived cultures, there was a greatvariability in the size and shape of the cells compared to the flaskculture. Diameter of the cell aggregation was enlarged up to 2-3 mm,which inhibited the flow inside of the bioreactor and developed unmixedregion in the bioreactor. Growth ring formed by the cells adhering tothe internal wall of the bioreactor. Cells in the center of the growthring died after 20 days because the media was not supplied efficiently.Eventually dead cells excreted toxic substances and these substanceslowered the vitality of all cells in the bioreactor. On the opposite,less aggregation of procambium-derived cultures caused smooth aircirculation in the bioreactor; hence it was possible to diminish theamount of air supply from 200 ml to 150 ml per minute and the amount ofdeveloped bubble on the surface of the media was greatly reduced.

Doubling time of the embryo and needle-derived cultures in the flask was12 days but it was lengthened to 21 days in the bioreactor. It wasbecause of the growth ring formation and rapid decrease of cellviability due to sensitiveness to shear by cell aggregation and rigidcell wall. Doubling time of procambium-derived cultures was 4 to 5 daysand there was no difference in the flask and the bioreactor, rather itwas shortened in the bioreactor (Table 4). Procambium-derived culturesformed very small growth ring in the bioreactor and the growth ring wasdissolved easily by agitating the media with a simple stimulus.Moreover, there was no decrease in cell viability due to lesssensitivity to shear by less cell aggregation and multiple vacuoles.

TABLE 4 Relationship between doubling time patterns and explants sourcein T. cuspidata cell cultures in flask and bioreactor Doubling time(day) Explant source flask bioreactor embryo 11.5 ± 1.3 21 ± 2.6 needle12 ± 2 21 ± 2   procambium   5 ± 0.2  4 ± 0.1

Practical Example 6 Elicitor

Elicitor controls molecular signal in plant cells and is widely used forthe increase of secondary metabolite productivity. After the treatmentof methyl jasmonate as an elicitor and 10 other kinds of elicitors, weobserved that methyl jasmonate had positive effect on the paclitaxelproduction. It was possible to obtain relatively high metabolitesproductivity through the combination of methyl jasmonate and otherelicitors. Especially, paclitaxel production was very effective with thetreatments of methyl jasmonate, chitosan and phenylanine (FIG. 4).

Practical Example 7 Conditioning Factors

Plant derived secondary metabolites are produced when the cells aregrowing or when the cells stopped growing. Therefore, two stage culturesare suitable for the production of metabolites like paclitaxel whosecell growth stage and metabolite production stage are separated. In thefirst stage, cells were proliferated in a large scale by optimizing thecell growth and in the second stage, the culture condition was changedfor the optimization of metabolites production.

Cell lines with high secondary metabolites productivity grow slower anddie faster than the cell lines with low productivity. Therefore, massproliferation is difficult and mass production of the metabolites isimpossible.

In this invention, cell lines with the ability of low proliferation andhigh production were not used for the proliferation in large scale,rather they were used as the helper cells that have the conditioningfactors for the production of secondary metabolites. We observed thepaclitaxel production after adding the helper cells. The results aresummarized in FIG. 5.

Practical Example 8 Perfusion Culture

On the day 14 of culture, elicitor was treated to the embryo andneedle-derived and procambium-derived cultures. From the point ofelicitation, spent media was retrieved in an aseptic condition withpipette on every 5 days and was supplied with the same amount of newmedia simultaneously. The production of paclitaxel in the cell and themedia were observed after 45 days of the long term culture. The resultwas summarized in Table 5.

TABLE 5 Paclitaxel production and release of T. cuspidata cells invarious explant sources and processes. Materials & Taxol yield (mg/kg)processes In cell In medium Total (days) Taxol release(%) embryo 12.97 ±1.16 0.03 ± 0.01 13 ± 1.17 (28) 0.2 ± 0.1 needle 10.92 ± 1.6  0.08 ±0.01 11 ± 1.6 (28)  0.7 ± 0.1 procambium 86.4 ± 6.7 32.3 ± 7.6  118.7 ±3.3 (28)    27.1 ± 6.1  procambium 0 0 0 (45) — Procambium 65.5 ± 4.1171.8 ± 11.1  237.3 ± 7 (45)     72.3 ± 2.5  perfusion culture

Depending on the cell lines, paclitaxel release of the cell to the mediawas different. Releasing ability of procambium-derived cultures wassuperior to the cultures of the preceding techniques. Theprocambium-derived single cell clone has an ability of releasing about404˜1077 times more paclitaxel than the cell lines derived from embryoand needle (in medium of Table 5). Moreover, application of perfusionculture facilitated the release of secondary metabolites to the media.Improvement in the extracellular release of secondary metabolitesthrough procambium-derived single cell clone by exchanging the mediaperiodically had great importance because it allowed continuous recycleof the biomass and simple purification.

In other words, periodical exchange of the media in theprocambium-derived single cell clone culture can be considered as astable method of producing valuable metabolites in the long termculture, because it prevents feedback inhibition of accumulatedmetabolites in the cell, degradation and conversion of the metabolitesin the media.

Practical Example 9 Cryopreservation

On the 6^(th) or 7^(th) day of the culture, suspension cells werepre-cultured in the media containing 0.16M of manitol for 3 days at theroom temperature and then maintained at 4° C. for 3 hours. Cells wereharvested and placed into 4 ml cryovial which had the media containing40% ethylene glycol (Sigma, USA) and 30% sorbitol (DUCHEFA, TheNetherlands) and cultured for 3 minutes at 4° C.

Suspension cells that were treated with cryopreservatives were frozenafter the cells were soaked in the liquid nitrogen. For thawing,cultured cells in the liquid nitrogen for more than 10 minutes werethawed in the 40° C. water bath for 1-2 minutes. For the re-growth ofthe cells, cryopreserved cells were transferred onto the semi-solidgrowth media (Table 1) containing 0.5 M sorbitol and alleviated at theroom temperature for 30 minutes. Cells were cultured on the semi-solidgrowth media containing 0.1M sorbitol for 24 hours. And then, the cellswere cultured on the semi-solid growth media without sorbitol for 24hours, twice. Cell viability was evaluated.

Practical Example 10 Analysis of Paclitaxel Content

After separating the cells from the media of the recovered samples,paclitaxel contents were analyzed. Cell mass was measured after dryingthe cells completely with vacuum desicator (Sam Shin Glass, Korea).About 100 mg (dry weight) of the cells were mixed with 4 ml solution(1:1 v/v) of methanol (Sigma, USA) and methylchloride (Sigma, USA) andwere extracted by ultrasonic cleaner (Branson, USA) for 3 times in onehour interval at the room temperature. Cells were fully dried andextracted several times by using 4 ml of methylchloride. Separatedorganic solvent layer was vacuum dried and the remaining was dissolvedin 1 ml of methanol. Dissolved extract was agitated equally byultrasonic cleaner. Then, after centrifugation, the pellet was removed(8,000 g×5 min).

Media (1-5 ml) that was separated from the cell was combined with thesame volume of methylchloride and was extracted 3 times after fullagitations. After organic solvent was vacuumed and dried completely, itwas dissolved in 0.5 ml of methanol again.

HPLC (High Performance Liquid Chromatography, Shiseido, Japan) was usedfor the analysis of the content and Sigma products were used forpaclitaxel standard substances. Capcell pak (C18, MGII, 5 um, 3.0 mm×250mm, Shiseido, Japan) was maintained to 40° C. by using the oven, andwater and acetonitril (Burdick & Jackson, USA) (50:50, v/v) werecombined for the mobile phase and dropped regularly with the speed of0.5 ml/min. UV-VIS detector (227 nm, Shiseido, Japan) was used.

INDUSTRIAL APPLICABILITY

In this invention, acquiring single cell clone, a primary meristem whichhas the meristematic continuity without dedifferentiation, by separatingprocambium purely from an apex of the twig resulted in higherproductivity due to shorter doubling time than the cell lines ofpreceding techniques. It also allowed stable productivity due to lesschange in the cell growth and growth pattern during the long termculture and scale up was possible because of less aggregation andmultiple vacuoles of the cell lines. This cell lines allowed recoveryafter cryopreservation without any genetic variation.

REFERENCES

-   1. Gamborg, O. L., Miller, R. A., Ojima, K. 1968. Nutrient    requirement of suspension cultures of soybean root cells. Exp. Cell.    Res., 50: 151-   2. Hirasuna T. J., Pestchanker L. J., Srinivasan V.,    Shuler M. L. (1996) Taxol production in suspension cultures of Taxus    baccata. Plant cell tissue and organ culture. 44:95-102-   3. Jouira H. B., Hassairi A., Bigot C., Dorion N. (1998)    Adventitious shoot production from strips of stem in the Dutch elm    hybrid ‘Commelin’: plantlet regeneration and neomycin sensitivity.    Plant cell Tissue and Organ Culture. 53:153-160-   4. Kim M. H., Chun S. H., Kim D. I. (2000) Growth promotion of Taxus    brevifolia cell suspension culture using conditioned medium.    Biotechnol bioprocess eng. 5:350-354-   5. Lloyd G. and McCown B. (1980) Commercially-feasible    micropropagation of mountain laurel, kalmia latifolia by use of    shoot-tip culture. Plant Prop. Proc. 30:421-   6. Muraghige T. and Skoog F. (1962) A revised medium for rapid    growth and bioassays with tobacco culture. Physiol Plant 15: 473-497-   7. Naill M. C., Roberts S. C. (2004) Preparation of single cells    from aggregated Taxus suspension cultures for population analysis.    Published online 10 May 2004 in wiley interscience.-   8. Quoirin M., Lepoivre P. (1977) Acta. Hart. 78: 437-   9. Schenk R. U., Hildebrandt A. C (1972) Medium and techniques for    induction and growth of monocotyledonous and dicotyledonous plant    cell culture. Can. J. Bot. 50: 199-   10. Wang C., Wu J., Mei X. (2001) Enhanced taxol production and    release in Taxus chininsis cell suspension cultures with selectid    organic solvents and sucrose feeding. Biotechnol. Prog. 17:89-94-   11. Wang C., Wu J., Mei X. (2001) Enhancement of taxol production    and excretion in Taxus chininsis cell culture by fungal elicitation    and medium renewal. Appl Microbiol Biotechnol. 55:404-410-   12. Wickremesinhe E. R. M., Arteca R. N. (1994) Taxus cell    suspension cultures: optimizing growth and production of taxol. J.    Plant Physiol. 144:183-188-   13. Wu J., Lin L. (2003) Enhancement of taxol production and release    in Taxus chinensis cell cultures by ultrasound, methyl jasmonate and    in situ solvent extraction. Appl Microbiol Biotechnol. 62:151-155-   14. Yokoi H., J. Koga, K. Yamamura and Y. Seike, (1993) High density    cultivation of plant cells in a new aeration-Agitation type    fermentor. 75:48-52-   15. Zhang C. H., Mei X. G., Liu L., Yu L. J. (2000) Enhanced    paclitaxel production induced by the combination of elicitors in    cell suspension cultures of Taxus chinensis. Biotechnology Letters.    22:1561-1564-   16. Zhong J. J. (2002) Plant cell culture for production of    paclitaxel and other taxanes. J. bioscience and bioengineering.    94:591-599

1. An isolated population of cells from a plant, wherein the cells ofthe isolated population are characterized in that they (i) are derivedfrom procambium of the plant, (ii) are innately undifferentiated, (iii)are homogeneous, and (iv) comprise the following characteristics: (a)including a greater number of single cells or including smaller-sizedcell aggregates in suspension culture than cells derived fromdedifferentiated callus of the plant; (b) having multiple vacuolesmorphologically; (c) being capable of growing faster and longer thancells derived from dedifferentiated callus of the plant; and (d) havinglower sensitivity to shear stress in a bioreactor than cells derivedfrom dedifferentiated callus of the plant.
 2. The isolated population ofcells according to claim 1, wherein the plant is the genus Taxus.
 3. Theisolated population of cells according to claim 2, wherein the cells arecapable of releasing paclitaxel 404-1077 times more than cells derivedfrom dedifferentiated callus of the plant.
 4. A method for producing abiologically active substance or substances, the method comprising thesteps of: (a) producing the active substance or substances by culturingin a medium the isolated population of cells of claim 1; and (b)collecting said active substance or substances.
 5. The method accordingto claim 4, wherein in the step (a), a predetermined amount of themedium that has been used to culture the cells is removed and apredetermined amount of a new medium is introduced.
 6. The methodaccording to claim 4, wherein the plant is the genus Taxus and theactive substance is paclitaxel.
 7. The method according to claim 6,wherein the medium includes at least one component selected from thegroup consisting of methyl jasmonate, phenylalanine, and chitosan.
 8. Amethod for preserving a plant cell line, the method comprisingcryopreserving the isolated population of cells of claim 1.